The molecular biology of mammalian arachidonic acid metabolism ELLIOTT SIGAL Cardiovascular Research Institute and Department University of California, San Francisco, California
of Medicine, 94 143-0911
SIGAL, FJLLIOTT. The molecular biology of mammalian arachidonic acid metabolism. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L13-LB, 1991.-The metabolism of arachidonic acid by cyclooxygenase and lipoxygenase enzymes results in a wide range of oxidized products with potent biological activities. These metabolites, which include the prostaglandins and leukotrienes, have been implicated in the pathogenesis of a variety of inflammatory diseases. Research over the last decade has focused primarily on the elucidation of the chemical structure of the metabolites and their biological effects in vitro and in vivo. Recently, research on the enzymes that produce these bioactive metabolites through oxidization of arachidonic acid has intensified. Recombinant DNA techniques have enabled investigators to determine the nucleotide sequences for several of the enzymes in the arachidonic acid cascade. The resulting cDNAs are now being used to further investigate the biochemical and biological features of arachidonic acid metabolism. The purpose of this paper is to review how the cDNAs for these enzymes were obtained, what information they convey, and how they are being applied in current research. CYClooxygen ase; lipoxygenase;
prostaglandin;
ACID is released from cell membranes by a variety of inflammatory stimuli and is oxidized to an array of compounds including the leukotrienes, the prostaglandins, and the thromboxanes. These biologically active metabolites have been implicated as critical mediators in inflammatory diseases such as bronchial asthma, arthritis, and psoriasis (33, 35, 47a, 85, 87). Consequently, the enzymes that form these oxidized derivatives of arachidonic acid have received considerable attention. Cyclooxygenase is the target enzyme for nonsteroidal anti-inflammatory agents such as aspirin. Lipoxygenases are current pharmacological targets for novel therapies in asthma. In recent years, recombinant DNA techniques have made possible the isolation of the cDNAs for several enzymes in the arachidonic acid cascade. These cDNAs have, in turn, been used to formulate and test new hypotheses about enzymatic function, to localize enzyme expression in disease, to delineate action of novel therapies, and to study gene expression. The aim of this paper is to review what has been learned in each of these areas. The focus here is on the mammalian cyclooxygenase and the three major lipoxygenase pathways. Studies on
ARACHIDONIC
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leukotriene
the related plant enzymes have greatly contributed to this field but are not a primary focus of this review (see Refs. 1,91,92, 121). Likewise, phospholipases and P-450 enzymes are relevant to arachidonic acid release and metabolism. For recent advances on the structures of phospholipases, see Refs. 43, 7589, 106. For the state of the art on P-450 enzymes, see Refs. 67, 68. This present review begins with a general overview of the arachidonic acid cascade. Following this is a discussion of how the cDNAs for the enzymes in the cascade were isolated and what information is provided by the deduced primary structures. Next, examples of how the cDNAs have been used to 1) analyze the mechanisms of drug actions, 2) probe expression of the enzymes in disease states, and 3) analyze enzymatic mechanisms at a molecular level are described. Finally, the use of the cDNAs to analyze gene expression is discussed. THE
ARACHIDONIC
ACID
CASCADE
Arachidonic acid is a 20-carbon fatty acid and it is a common constituent of phospholipids in cell membranes. On stimulation of a cell, free arachidonic acid is released from the cell membrane by the action of phospholipases.
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Physiological
Society
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The signals that cause the release of arachidonic acid are not fully understood but appear to include a variety of receptors coupled by G proteins to a set of second messengers. Stimulation of the receptor and consequent signal transduction results in mobilization of calcium and activation of phospholipases (11). The phospholipases release free arachidon ic acid which can then be oxidized by several pathways. There is considerable variety in the potential metabolic pathways for arachidonic acid within the body; however, specific cells are relatively selective in the oxidation of this fatty acid. For detailed reviews of the biology and biochemistry see of the metabolites, Refs. 37, 44, 47a, 62, 66, 85, 87. Once arachidonic acid is released from cell membranes, it may be metabolized via the cyclooxygenase pathway or via the lipoxygenase pathway. Figure 1 displays an overview of arachidonic acid metabolism. The release of arachidonic acid from the membrane appears to be inhibited by steroids (ZZ), although the mechanism has been recently questioned (2, 10) The biotransformation of free arachi donic acid via the’ enzyme cyclooxygenase leads to the formation of prostaglandin (PG) DP, PGE2, and PGFZty as well as the formation of thromboxane A, and prostacyclin. Cyclooxygenase is inhibited by aspirin
REVIEW
and other nonsteroidal anti-inflammatory agents. Cyclooxygenase catalyzes the attachment of molecular oxygen at the 11th carbon of arachidonic acid. There is a subsequent rearrangement to a cyclic endoperoxide in which a molecule of oxygen bridges carbons 9 and 11. An introduction of a second molecule of oxygen at carbon 15 yields PGG2. The subscript in prostaglandin nomenclature refers to the two double bonds in the structure. Hydroperoxidase activity (also performed by cyclooxygenase) leads to the formation of PGH, from PGG,. Thromboxane AZ, the predominant product of arachidonic acid metabolism from platelets, is formed from PGH2 and is a potent aggregating agent. In addition, thromboxane A2 is a vasoconstrictor and a bronchoconstrictor. For a select list of biological actions associated with various metabolites, see Table 1. Prostacyclin, which is also derived from the formation of PGHZ, is a potent inhibitor of platelet aggregation and in some vascular systems is thought to antagonize the formation of thromboxane A2 and serve as a vasodilator. The prostaglandin synthases D, E, and F convert PGH, to PGD2, PGE2, PGF2,,, respectively. Prostaglandin D2 is formed from mast cells upon antigen binding of IgE receptors. Like histamine, PGD2 is a potent bronchoconstrictor. Prostacyl Thrombox Prostaglandin
11
vPGH2
Prostaglandln
’
tin ane
A 2 Di,E
z,F
2a
Synthetases aLeukotriene
G2
64
Aspirin l-l-
Cyclooxygenase
LTA4
S-HPETE
Leukotriene
C4,D
4 ,E 4
S-LO inhibitors
(SRS-A) Phospholipases Membrane Phospholiplds
Steriods
15-HETE Lipoxins 8,15 diHETE 15-HPETE
12-Lipoxygenase
-
-
12-HETE
12-HPETE
Arachidonic acid metabolism via cyclooxygenase and lipoxygenase (LO) pathways. Putative sites ot action f’or steroids, aspirin, and 5-LO inhibitors are indicated by shaded boxes. Cyclooxygenase performs bisoxygenation of arachidonic acid to form prostaglandin (PG) G,. PGG, is reduced by cyclooxygenase to PGH,. PGH, is converted by various synthetases to either prostacyclin, thromboxanes, or PGD2, Ez, or Fz,,. Lipoxygenases insert molecular oxygen at carbon 5, 15, or 12 of arachidonic acid thereby forming 5, 15, or 12-hydroperoxyeicosatetraenoic acids (HPETEs). These HPETEs are further metabolized to leukotrienes (LT), mono- or di-HETEs or lipoxins. The slow-reacting substance of anaphylaxis (SRS-A) is a mixture of LTC4, D4, and E4. FIG.
1.
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INVITED TABLE
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1. Selected arachidonic acid metabolites and a partial list of biological activities Enzymes Involved in Synthesis
Metabolite
PGD, PCJE,
CO, PGD CO, PGE
synthase synthase
PGF,,,
CO, PGE
synthase
Thromboxane
A,
Prostacyclin
Leukot
riene
Leukotriene (SRS-A)
CO, PGI
5-LO,
C4, D4, E.,
5-LO, LTC synthase peptidases)
CO, cyclooxygenase; not been established.
5-LO, 5-LO
A,
synthase
B.%
HETEs Lipoxins LO, lipoxygenase; biological roles have
CO, thromboxane synt hase
Major
LTA,
hydrolase (plus
n-Lo, 15-LO and 15-LO
Biological Activity
Bronchoconstriction Bronchodilation, vasodilation, increase in epithelial chloride secretion Bronchoconstriction, vasoconstriction, increase in epithelial chloride secretion Bronchoconstriction, vasoconstriction, platelet aggregation Vasodilation, increase in vascular permeability, inhibition of platelet aggregation Leukocyte migration, adhesion, and activation Bronchoconstriction, vasoconstriction, increase in vascular permeability * *
PG, prostaglandin; LT, leukotriene. * Multiple biological activities For a review on HETEs see Ref. 102 and for lipoxins see Ref. 87.
Prostaglandin E, is a vasodilator and a bronchodilator, whereas PGFZ,, is a vasoconstrictor and bronchoconstrictor. Human airway epithelial cells have been shown to produce both PGE, and PGFZ,, in response to inflammatory stimuli (117), and these mediators act to increase chloride secretion (18,117). These findings are important because airway- epithelial cells interface with the external environment where common inflammatory stimuli originate. An alternate pathway of oxidation of arachidonic acid 1s through the lipoxygenase pathway. There are three major lipoxygenases named for their ability to insert molecular oxygen at a specific carbon of arachidonic acid (Fig. 1). The 5-lipoxygenase initiates the generation of leukotrienes by forming a hydroperoxy fatty acid (5HPETE) and another intermediate, leukotriene (LT) A,. The enzyme LTA4 hydrolase metabolizes LTA, to LTB, in neutrophils and macrophages. LTB, is the most potent chemoattractant for leukocytes and causes adherence and chemotaxis of leukocytes at nanomolar concentrations (24). Alternatively, LTA, can be metabolized to LT&, LTD4, and LTE+ which constitute the mixture previously called “slow-reacting substance of anaphylaxis” (SRS-A) (57). These leukotrienes are synthesized primarily by mast cells, eosinophils, and macrophages and are potent bronchoconstrictors (9). These compounds also increase microvascular permeability and induce airway secretion. The biological roles of the 12- and 15-lipoxygenases are less clear. The action of both enzymes results in an array of hydroxy eicosatetraenoic acids (HETEs). For a review of the biological activities of HETEs, see Ref. 102. 12-Lipoxygenase predominates in human platelets, and one potential role of its product, 12-HETE, is the modulation of adhesion receptors (30). The 15-lipoxygenase is detected in developing red cells (73), in human eosinophils (1 ll), and in airway epithelial cells (34, 40). Metabolites generated from 15lipoxygenation of arachidonic acid include 15-HETE, dihydroxy acids (e.g., 8,15-
have
been
described
but
firm
diHETE), and trihydroxy acids called lipoxins. Lipoxins may also result from sequential action of 5-, 12 or 15lipoxygenase. Products of 15lipoxygenase have been detected in human bronchoalveolar lavage (46,58) and may contribute to airway inflammation (42, 90), secretion (41, 50) and the modulation of C-fiber responses (7, 47, 116). The 15-lipoxygenase may act on substrates other than arachidonic acid and has been implicated in cell differentiation (45, 73) and the inflammatory response associated with atherosclerosis ( 105, 122). In summary, the metabolites of arachidonic acid can evoke all of the cardinal signs of inflammation by eliciting vasodilation, increased vascular permeability, pain, and the influx of leukocytes. In the airways, relevant cells such as eosinophils, mast cells, and macrophages release leukotrienes that result in bronchoconstriction. For these reasons, and because the pathway is a site of action for steroids, aspirin, and other nonsteroidal antiinflammatory agents, the metabolism of arachidonic acid has received increasing attention. Research in the previous two decades has focused on the chemical elucidation of the structure of arachidonic acid metabolites and on testing their biological activity in vitro and in vivo. Over the last decade, there has been increasing emphasis on the enzymes that initiate the oxidation of arachidonic acid. The purification and characterization of many of these enzymes have been achieved. An increasing number of investigations are now focused on the molecular structure of the enzymes in the pathway. The cDNAs for several of these enzymes have been isolated and are being used as reagents for the molecular and biological study of arachidonic acid metabolism. The following sections highlight some of the features of this current research. ISOLATION OF ACID SEQUENCE:
cDNAs THE
AND PREDICTIONS INITIAL STEPS
OF
AMINO
The cDNAs encoding cyclooxygenase, PGD synthase, PGF synthase, &lipoxygenase, LTA4 hydrolase, 15-li-
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poxygenase, and 12lipoxygenase have been isolated and characterized. In general, the cloning strategies have relied on the previous isolation of protein and either corresponding antibodies or oligonucleotides as probes to screen cDNA libraries. An exception has been the recent cloning of human platelet 12lipoxygenase, which used the polymerase chain reaction and homologous lipoxygenase sequences to obtain probes for a protein that has not yet been purified. In all cases, information on the primary structure of the enzymes has provided evidence of their evolutionary origin and hypotheses that relate structural features to enzymatic functions. This section reviews the cloning strategies and the initial information about enzyme structure that has emerged from the cDNAs for cyclooxygenase, the prostaglandin synthases, LTA, hydrolase, and the three major lipoxygenases.
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EPIDERMAL DOMAIN GLYCOSYLATION
SITES
mmmmmm
mm
(Prostaglandin
FACTOR
MEMBRANE mmmmmm
mm
mmmm
PEROXIDASE CYTOPLASMIC
Cyclooxygenase
GROWTH
DOMAIN
DOMAIN
G/H Synthase)
Cyclooxygenase (also called prostaglandin G/H synthase) is the first enzyme in the pathway of arachidonic acid metabolism that produces prostacyclin, thromboxane AZ, PGDZ, PGEZ, and PGFaty. The enzyme is bound to heme and performs both a dioxygenase function to form PGGSTEIN. A novel dioxygenat,ion product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes. J. Biol. Chem. 258: 14948-1495:3, 1983. 91. SHIBATA, D., .J. STECZKO, J. E. DIXON, P. C. ANI~REWS, M. HERMODSON, AND B. AXEI,ROD. Primary structure of soybean lipoxygenase L-2. J. Biol. Chem. 26:3: 6816-6821, 1988. 92. SHIBATA, D., J. STECZKO, J. E. DIXON, M. HERMODSON, R. YAZI~ANPARAST, AND B. AXELROD. Primary structure of soybean lipoxygenase-1. J. Biol. Chum. 262: 10080-10085, 1987. 93. SHIMOKAWA, T., D. L. DEWITT, AND W. L. SMITH. The aspirin and heme binding sit,es of PGH synthase. In: Advances in Prostaglandin, Thromboxane, and Leukotriene Research, edited by B. Samuelsson, R. Paolet,ti, and P. W. Ramwell. New York: Raven, vol. 21. 94. SXAL, E., C. S. CRAIK, E. HIC.HI,ANI), D. GRCJNBER(;ER, L. L. COSTELI,O, R. A. F. DIXON, AND J. A. NADEL. Molecular cloning and primary st,ructure of human UXpoxygenase. B&hem. Biophys. Res. Commun. 157: 457-464, 1988. 95. SI(;AL, E., D. GRUNBERGER, J. R. CASHMAN, C. S. CRAIK, G. H. CAUGHEY, ANI) J. A. NADEL. Arachidonate 15lipoxygenase from human eosinophil-enriched leukocytes: partial purification and propert.ies. Biochem. Biophys. Res. Commun. 150: 376-38:3, 1988. 96. SIC;AL, E., D. GRUNBERC.ER, C. S. CRAIK, G. H. CAIJGHEY, AND J. A. NAIIEI,. Arachidonate 15lipoxygenase (w-6 lipoxygenase) from human leukocyt,es: purification and st,ructural homology to other mammalian lipoxygenases. J. Biol. Chem. 26:3: E&28-X3X2, 1988. 97. SIGAI,, E., D. GRUNBERGER, E. HEHLANI), C. GROSS, R. A. F. DIXON, AND C. S. CRAIK. Expression of cloned human reticulocyte 15lipoxygenase and immunological evidence t.hat, lfi-lipoxygenases of different cell types are related. J. Biol. Chem. 265: 511% 5120, 1990. 98. SI,OANE, D., R. A. F. DIXON, (1. S. CRAIK, AND E. SI~:AI,. Expression of’ cloned human Ifi-lipoxygenase in eukaryotic and prokaryot,ic systems. In: Advances in Prostaglandin, 7’hromboxane, and Leukotriene Research, edited by B. Samuelsson. New York: Raven. 99. SLOANE, D. L., M. F. BROWNER, Z. DAWTER, K. WILSON, R. J. FI,ETTERICK, AND E. SIGAL. Purification and crystallization of 15lipoxygenase from rabbit reticulocytes. Hiochem. Biophys. Rcs. Commun. In press. 100. SLOANE, D. L., C. S. CRAIK, AND E. SI(;AI,. The expression of active human reticulocyt,e 15lipoxygenase in bact,eria. Biomed. B&him. Acta 49: Sll-S16, 1990. 101. SMITH, W. L., D. L. DEWITT, S. A. KRAEMER, M. ,J. ANDREWS, T. HLA, T. MACIA(;, AND T. SHIMOKAWA. Structure-function relationships in sheep, mouse, and human prost,aglandin endoperoxide G/H synt hases. In: Advances in Prostaglandin, Thromboxane, and Leukotriene Research, edited by B. Samuelsson, S.-E. Dahlen, ,J. Fritsch, and P. Hedqvist. New York: Raven, vol. 20, p. 14. 102. SPECTOR, A. A., J. A. GORIION, ANI) S. A. MOORE. Hydroxyeicosatetraenoic acids (HETEs). Prog. Lipid. Res. 27: 271-22~3, 1988. 103. STALI,IN(;S, W. C., B. A. KROA, R. T. CARROX, A. L. METZ(:ER, AND M. 0. FIJNK. Crystallization and preliminary x-ray characterization of a soybean seed lipoxygenase. J. Mol. Biol. 211: 68% 687, 1990. 104. STECZKO, , .J., C. R. MUCI~MORE, J. L. SMITH, ANI) B. AXEI,ROI).
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INVITED Crystallization and preliminary x-ray investigation of lipoxygenase 1 from soybeans. J. Biol. Chem. 265: 11352-11354, 1990. STEINBERG, D., S. PARTHASARATHY, T. E. CAREW, J. C. KHOO, AND J. L. WITZTUM. Beyond cholesterol: modifications of lowdensity lipoprotein that increase its atherogenicity. N. En& J. Med. 320: 915-924, 1989. SUH, P.-G., S. H. RYU, K. H. MOON, H. W. SUH, AND S. G. RHEE. Cloning and sequence of multiple forms of phospholipase C. Cell 54: 161-169, 1988. TAKAHASHI, Y., N. UEDA, AND S. YAMAMOTO. TWO immunologically and catalytically distinct arachidonate 12-lipoxygenases of bovine platelets and leukocytes. Arch. Biochem. Biophys. 266: 613-621, 1988. THIELE, B. J., M. HOHNE, B. NACK, P. R. HARRISON, AND S. M. RAPOPORT. Lipoxygenase mRNA during development of red blood cells studied with a cloned probe. Biomed. Biochim. Acta 46: S124-s125, 1987. THIELE, J., H. ANDREE, M. HOHNE, AND S. M. RAPOPORT. Lipoxygenase mRNA in rabbit reticulocytes. Its isolation, characterization and translational repression. Eur. J. Biochem. 129: 133-141, 1982. TOH, H. Prostaglandin endoperoxide synthase contains an EGFlike domain. FEBS Lett. 258: 317-319, 1989. TURK, J., R. L. MASS, A. R. BRASH, L. J. ROBERTS, II, AND J. A. OATES. Arachidonic acid 15-lipoxygenase products from human eosinophils. J. Biol. Chem. 257: 7068-7076, 1982. URADE, Y., A. NACATA, Y. SUZUKI, Y. RUJII, AND 0. HAYAISHI. Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence. J. Biol. Chem. 264: 1041-1045,1989. VANE, J. R. Inhibition of prostaglandin synthesis as a mechanism of action for the aspirin-like drugs. Nature Lond. 231: 232-235, 1971. WATANABE, K., Y. FUJII, H. HAYASHI, Y. URADE, AND 0. HAYAISHI. The structure and function of prostaglandin F synthetase. In: Advances in Prostaglandin, Thromboxane, and Leukotriene Research, edited by B. Samuelsson, R. Paoletti, and P. W. Ramwell. New York: Raven, vol. 21. WATANABE, K., F. YUTAKA, K. NAKAYAMA, H. OHKUBO, S. KuRAMITSU, H. KAGAMIYAMA, S. NAKANISHI, AND 0. HAYAISHI. Structural similarity of bovine lung prostaglandin F synthase to lens f-crystallin of the European common frog. Proc. N&l. Acad. Sci. IJSA 85: 11-15, 1988. WHITE, D. M., A. I. BASBAUM, E. J. GOETZL, AND J. D. LEVINE. The 15-lipoxygenase product, 8R,15S-diHETE, stereospecifically sensitizes C-fiber mechanoheat nociceptors in hairy skin of rat. J. Neurophysiol. 63: 966-970, 1990. WIDDICOMBE, J. H., I. F. UEKI, D. EMERY, D. MARGOLSKEE, J.
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of Medicine, 94 143-0911
SIGAL, FJLLIOTT. The molecular biology of mammalian arachidonic acid metabolism. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4): L13-LB, 1991.-The metabolism of arachidonic acid by cyclooxygenase and lipoxygenase enzymes results in a wide range of oxidized products with potent biological activities. These metabolites, which include the prostaglandins and leukotrienes, have been implicated in the pathogenesis of a variety of inflammatory diseases. Research over the last decade has focused primarily on the elucidation of the chemical structure of the metabolites and their biological effects in vitro and in vivo. Recently, research on the enzymes that produce these bioactive metabolites through oxidization of arachidonic acid has intensified. Recombinant DNA techniques have enabled investigators to determine the nucleotide sequences for several of the enzymes in the arachidonic acid cascade. The resulting cDNAs are now being used to further investigate the biochemical and biological features of arachidonic acid metabolism. The purpose of this paper is to review how the cDNAs for these enzymes were obtained, what information they convey, and how they are being applied in current research. CYClooxygen ase; lipoxygenase;
prostaglandin;
ACID is released from cell membranes by a variety of inflammatory stimuli and is oxidized to an array of compounds including the leukotrienes, the prostaglandins, and the thromboxanes. These biologically active metabolites have been implicated as critical mediators in inflammatory diseases such as bronchial asthma, arthritis, and psoriasis (33, 35, 47a, 85, 87). Consequently, the enzymes that form these oxidized derivatives of arachidonic acid have received considerable attention. Cyclooxygenase is the target enzyme for nonsteroidal anti-inflammatory agents such as aspirin. Lipoxygenases are current pharmacological targets for novel therapies in asthma. In recent years, recombinant DNA techniques have made possible the isolation of the cDNAs for several enzymes in the arachidonic acid cascade. These cDNAs have, in turn, been used to formulate and test new hypotheses about enzymatic function, to localize enzyme expression in disease, to delineate action of novel therapies, and to study gene expression. The aim of this paper is to review what has been learned in each of these areas. The focus here is on the mammalian cyclooxygenase and the three major lipoxygenase pathways. Studies on
ARACHIDONIC
1040-0605/91
$1.50
Copyright
leukotriene
the related plant enzymes have greatly contributed to this field but are not a primary focus of this review (see Refs. 1,91,92, 121). Likewise, phospholipases and P-450 enzymes are relevant to arachidonic acid release and metabolism. For recent advances on the structures of phospholipases, see Refs. 43, 7589, 106. For the state of the art on P-450 enzymes, see Refs. 67, 68. This present review begins with a general overview of the arachidonic acid cascade. Following this is a discussion of how the cDNAs for the enzymes in the cascade were isolated and what information is provided by the deduced primary structures. Next, examples of how the cDNAs have been used to 1) analyze the mechanisms of drug actions, 2) probe expression of the enzymes in disease states, and 3) analyze enzymatic mechanisms at a molecular level are described. Finally, the use of the cDNAs to analyze gene expression is discussed. THE
ARACHIDONIC
ACID
CASCADE
Arachidonic acid is a 20-carbon fatty acid and it is a common constituent of phospholipids in cell membranes. On stimulation of a cell, free arachidonic acid is released from the cell membrane by the action of phospholipases.
!G 1991 the American
Physiological
Society
L13
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INVITED
The signals that cause the release of arachidonic acid are not fully understood but appear to include a variety of receptors coupled by G proteins to a set of second messengers. Stimulation of the receptor and consequent signal transduction results in mobilization of calcium and activation of phospholipases (11). The phospholipases release free arachidon ic acid which can then be oxidized by several pathways. There is considerable variety in the potential metabolic pathways for arachidonic acid within the body; however, specific cells are relatively selective in the oxidation of this fatty acid. For detailed reviews of the biology and biochemistry see of the metabolites, Refs. 37, 44, 47a, 62, 66, 85, 87. Once arachidonic acid is released from cell membranes, it may be metabolized via the cyclooxygenase pathway or via the lipoxygenase pathway. Figure 1 displays an overview of arachidonic acid metabolism. The release of arachidonic acid from the membrane appears to be inhibited by steroids (ZZ), although the mechanism has been recently questioned (2, 10) The biotransformation of free arachi donic acid via the’ enzyme cyclooxygenase leads to the formation of prostaglandin (PG) DP, PGE2, and PGFZty as well as the formation of thromboxane A, and prostacyclin. Cyclooxygenase is inhibited by aspirin
REVIEW
and other nonsteroidal anti-inflammatory agents. Cyclooxygenase catalyzes the attachment of molecular oxygen at the 11th carbon of arachidonic acid. There is a subsequent rearrangement to a cyclic endoperoxide in which a molecule of oxygen bridges carbons 9 and 11. An introduction of a second molecule of oxygen at carbon 15 yields PGG2. The subscript in prostaglandin nomenclature refers to the two double bonds in the structure. Hydroperoxidase activity (also performed by cyclooxygenase) leads to the formation of PGH, from PGG,. Thromboxane AZ, the predominant product of arachidonic acid metabolism from platelets, is formed from PGH2 and is a potent aggregating agent. In addition, thromboxane A2 is a vasoconstrictor and a bronchoconstrictor. For a select list of biological actions associated with various metabolites, see Table 1. Prostacyclin, which is also derived from the formation of PGHZ, is a potent inhibitor of platelet aggregation and in some vascular systems is thought to antagonize the formation of thromboxane A2 and serve as a vasodilator. The prostaglandin synthases D, E, and F convert PGH, to PGD2, PGE2, PGF2,,, respectively. Prostaglandin D2 is formed from mast cells upon antigen binding of IgE receptors. Like histamine, PGD2 is a potent bronchoconstrictor. Prostacyl Thrombox Prostaglandin
11
vPGH2
Prostaglandln
’
tin ane
A 2 Di,E
z,F
2a
Synthetases aLeukotriene
G2
64
Aspirin l-l-
Cyclooxygenase
LTA4
S-HPETE
Leukotriene
C4,D
4 ,E 4
S-LO inhibitors
(SRS-A) Phospholipases Membrane Phospholiplds
Steriods
15-HETE Lipoxins 8,15 diHETE 15-HPETE
12-Lipoxygenase
-
-
12-HETE
12-HPETE
Arachidonic acid metabolism via cyclooxygenase and lipoxygenase (LO) pathways. Putative sites ot action f’or steroids, aspirin, and 5-LO inhibitors are indicated by shaded boxes. Cyclooxygenase performs bisoxygenation of arachidonic acid to form prostaglandin (PG) G,. PGG, is reduced by cyclooxygenase to PGH,. PGH, is converted by various synthetases to either prostacyclin, thromboxanes, or PGD2, Ez, or Fz,,. Lipoxygenases insert molecular oxygen at carbon 5, 15, or 12 of arachidonic acid thereby forming 5, 15, or 12-hydroperoxyeicosatetraenoic acids (HPETEs). These HPETEs are further metabolized to leukotrienes (LT), mono- or di-HETEs or lipoxins. The slow-reacting substance of anaphylaxis (SRS-A) is a mixture of LTC4, D4, and E4. FIG.
1.
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INVITED TABLE
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REVIEW
1. Selected arachidonic acid metabolites and a partial list of biological activities Enzymes Involved in Synthesis
Metabolite
PGD, PCJE,
CO, PGD CO, PGE
synthase synthase
PGF,,,
CO, PGE
synthase
Thromboxane
A,
Prostacyclin
Leukot
riene
Leukotriene (SRS-A)
CO, PGI
5-LO,
C4, D4, E.,
5-LO, LTC synthase peptidases)
CO, cyclooxygenase; not been established.
5-LO, 5-LO
A,
synthase
B.%
HETEs Lipoxins LO, lipoxygenase; biological roles have
CO, thromboxane synt hase
Major
LTA,
hydrolase (plus
n-Lo, 15-LO and 15-LO
Biological Activity
Bronchoconstriction Bronchodilation, vasodilation, increase in epithelial chloride secretion Bronchoconstriction, vasoconstriction, increase in epithelial chloride secretion Bronchoconstriction, vasoconstriction, platelet aggregation Vasodilation, increase in vascular permeability, inhibition of platelet aggregation Leukocyte migration, adhesion, and activation Bronchoconstriction, vasoconstriction, increase in vascular permeability * *
PG, prostaglandin; LT, leukotriene. * Multiple biological activities For a review on HETEs see Ref. 102 and for lipoxins see Ref. 87.
Prostaglandin E, is a vasodilator and a bronchodilator, whereas PGFZ,, is a vasoconstrictor and bronchoconstrictor. Human airway epithelial cells have been shown to produce both PGE, and PGFZ,, in response to inflammatory stimuli (117), and these mediators act to increase chloride secretion (18,117). These findings are important because airway- epithelial cells interface with the external environment where common inflammatory stimuli originate. An alternate pathway of oxidation of arachidonic acid 1s through the lipoxygenase pathway. There are three major lipoxygenases named for their ability to insert molecular oxygen at a specific carbon of arachidonic acid (Fig. 1). The 5-lipoxygenase initiates the generation of leukotrienes by forming a hydroperoxy fatty acid (5HPETE) and another intermediate, leukotriene (LT) A,. The enzyme LTA4 hydrolase metabolizes LTA, to LTB, in neutrophils and macrophages. LTB, is the most potent chemoattractant for leukocytes and causes adherence and chemotaxis of leukocytes at nanomolar concentrations (24). Alternatively, LTA, can be metabolized to LT&, LTD4, and LTE+ which constitute the mixture previously called “slow-reacting substance of anaphylaxis” (SRS-A) (57). These leukotrienes are synthesized primarily by mast cells, eosinophils, and macrophages and are potent bronchoconstrictors (9). These compounds also increase microvascular permeability and induce airway secretion. The biological roles of the 12- and 15-lipoxygenases are less clear. The action of both enzymes results in an array of hydroxy eicosatetraenoic acids (HETEs). For a review of the biological activities of HETEs, see Ref. 102. 12-Lipoxygenase predominates in human platelets, and one potential role of its product, 12-HETE, is the modulation of adhesion receptors (30). The 15-lipoxygenase is detected in developing red cells (73), in human eosinophils (1 ll), and in airway epithelial cells (34, 40). Metabolites generated from 15lipoxygenation of arachidonic acid include 15-HETE, dihydroxy acids (e.g., 8,15-
have
been
described
but
firm
diHETE), and trihydroxy acids called lipoxins. Lipoxins may also result from sequential action of 5-, 12 or 15lipoxygenase. Products of 15lipoxygenase have been detected in human bronchoalveolar lavage (46,58) and may contribute to airway inflammation (42, 90), secretion (41, 50) and the modulation of C-fiber responses (7, 47, 116). The 15-lipoxygenase may act on substrates other than arachidonic acid and has been implicated in cell differentiation (45, 73) and the inflammatory response associated with atherosclerosis ( 105, 122). In summary, the metabolites of arachidonic acid can evoke all of the cardinal signs of inflammation by eliciting vasodilation, increased vascular permeability, pain, and the influx of leukocytes. In the airways, relevant cells such as eosinophils, mast cells, and macrophages release leukotrienes that result in bronchoconstriction. For these reasons, and because the pathway is a site of action for steroids, aspirin, and other nonsteroidal antiinflammatory agents, the metabolism of arachidonic acid has received increasing attention. Research in the previous two decades has focused on the chemical elucidation of the structure of arachidonic acid metabolites and on testing their biological activity in vitro and in vivo. Over the last decade, there has been increasing emphasis on the enzymes that initiate the oxidation of arachidonic acid. The purification and characterization of many of these enzymes have been achieved. An increasing number of investigations are now focused on the molecular structure of the enzymes in the pathway. The cDNAs for several of these enzymes have been isolated and are being used as reagents for the molecular and biological study of arachidonic acid metabolism. The following sections highlight some of the features of this current research. ISOLATION OF ACID SEQUENCE:
cDNAs THE
AND PREDICTIONS INITIAL STEPS
OF
AMINO
The cDNAs encoding cyclooxygenase, PGD synthase, PGF synthase, &lipoxygenase, LTA4 hydrolase, 15-li-
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poxygenase, and 12lipoxygenase have been isolated and characterized. In general, the cloning strategies have relied on the previous isolation of protein and either corresponding antibodies or oligonucleotides as probes to screen cDNA libraries. An exception has been the recent cloning of human platelet 12lipoxygenase, which used the polymerase chain reaction and homologous lipoxygenase sequences to obtain probes for a protein that has not yet been purified. In all cases, information on the primary structure of the enzymes has provided evidence of their evolutionary origin and hypotheses that relate structural features to enzymatic functions. This section reviews the cloning strategies and the initial information about enzyme structure that has emerged from the cDNAs for cyclooxygenase, the prostaglandin synthases, LTA, hydrolase, and the three major lipoxygenases.
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EPIDERMAL DOMAIN GLYCOSYLATION
SITES
mmmmmm
mm
(Prostaglandin
FACTOR
MEMBRANE mmmmmm
mm
mmmm
PEROXIDASE CYTOPLASMIC
Cyclooxygenase
GROWTH
DOMAIN
DOMAIN
G/H Synthase)
Cyclooxygenase (also called prostaglandin G/H synthase) is the first enzyme in the pathway of arachidonic acid metabolism that produces prostacyclin, thromboxane AZ, PGDZ, PGEZ, and PGFaty. The enzyme is bound to heme and performs both a dioxygenase function to form PGGSTEIN. A novel dioxygenat,ion product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes. J. Biol. Chem. 258: 14948-1495:3, 1983. 91. SHIBATA, D., .J. STECZKO, J. E. DIXON, P. C. ANI~REWS, M. HERMODSON, AND B. AXEI,ROD. Primary structure of soybean lipoxygenase L-2. J. Biol. Chem. 26:3: 6816-6821, 1988. 92. SHIBATA, D., J. STECZKO, J. E. DIXON, M. HERMODSON, R. YAZI~ANPARAST, AND B. AXELROD. Primary structure of soybean lipoxygenase-1. J. Biol. Chum. 262: 10080-10085, 1987. 93. SHIMOKAWA, T., D. L. DEWITT, AND W. L. SMITH. The aspirin and heme binding sit,es of PGH synthase. In: Advances in Prostaglandin, Thromboxane, and Leukotriene Research, edited by B. Samuelsson, R. Paolet,ti, and P. W. Ramwell. New York: Raven, vol. 21. 94. SXAL, E., C. S. CRAIK, E. HIC.HI,ANI), D. GRCJNBER(;ER, L. L. COSTELI,O, R. A. F. DIXON, AND J. A. NADEL. Molecular cloning and primary st,ructure of human UXpoxygenase. B&hem. Biophys. Res. Commun. 157: 457-464, 1988. 95. SI(;AL, E., D. GRUNBERGER, J. R. CASHMAN, C. S. CRAIK, G. H. CAUGHEY, ANI) J. A. NADEL. Arachidonate 15lipoxygenase from human eosinophil-enriched leukocytes: partial purification and propert.ies. Biochem. Biophys. Res. Commun. 150: 376-38:3, 1988. 96. SIC;AL, E., D. GRUNBERC.ER, C. S. CRAIK, G. H. CAIJGHEY, AND J. A. NAIIEI,. Arachidonate 15lipoxygenase (w-6 lipoxygenase) from human leukocyt,es: purification and st,ructural homology to other mammalian lipoxygenases. J. Biol. Chem. 26:3: E&28-X3X2, 1988. 97. SIGAI,, E., D. GRUNBERGER, E. HEHLANI), C. GROSS, R. A. F. DIXON, AND C. S. CRAIK. Expression of cloned human reticulocyte 15lipoxygenase and immunological evidence t.hat, lfi-lipoxygenases of different cell types are related. J. Biol. Chem. 265: 511% 5120, 1990. 98. SI,OANE, D., R. A. F. DIXON, (1. S. CRAIK, AND E. SI~:AI,. Expression of’ cloned human Ifi-lipoxygenase in eukaryotic and prokaryot,ic systems. In: Advances in Prostaglandin, 7’hromboxane, and Leukotriene Research, edited by B. Samuelsson. New York: Raven. 99. SLOANE, D. L., M. F. BROWNER, Z. DAWTER, K. WILSON, R. J. FI,ETTERICK, AND E. SIGAL. Purification and crystallization of 15lipoxygenase from rabbit reticulocytes. Hiochem. Biophys. Rcs. Commun. In press. 100. SLOANE, D. L., C. S. CRAIK, AND E. SI(;AI,. The expression of active human reticulocyt,e 15lipoxygenase in bact,eria. Biomed. B&him. Acta 49: Sll-S16, 1990. 101. SMITH, W. L., D. L. DEWITT, S. A. KRAEMER, M. ,J. ANDREWS, T. HLA, T. MACIA(;, AND T. SHIMOKAWA. Structure-function relationships in sheep, mouse, and human prost,aglandin endoperoxide G/H synt hases. In: Advances in Prostaglandin, Thromboxane, and Leukotriene Research, edited by B. Samuelsson, S.-E. Dahlen, ,J. Fritsch, and P. Hedqvist. New York: Raven, vol. 20, p. 14. 102. SPECTOR, A. A., J. A. GORIION, ANI) S. A. MOORE. Hydroxyeicosatetraenoic acids (HETEs). Prog. Lipid. Res. 27: 271-22~3, 1988. 103. STALI,IN(;S, W. C., B. A. KROA, R. T. CARROX, A. L. METZ(:ER, AND M. 0. FIJNK. Crystallization and preliminary x-ray characterization of a soybean seed lipoxygenase. J. Mol. Biol. 211: 68% 687, 1990. 104. STECZKO, , .J., C. R. MUCI~MORE, J. L. SMITH, ANI) B. AXEI,ROI).
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